Yttrium-doped zirconia bicrystals with [001] symmetric tilt Sigma 5 grain boundaries were fabricated by a diffusion bonding technique, and oxygen diffusion behavior across the grain boundary was measured by secondary ion mass spectrometry (SIMS), tracing O-18 isotope. It was found that the O-18 fraction across the boundary exhibited explicit decrease around the boundary plane, indicating that the oxygen-diffusion is blocked by the presence of the Sigma 5 grain boundary. This is the first experimental detection of oxygen diffusion blocking at a single grain boundary in zirconia ceramics. From high-resolution transmission electron microscopy observations and energy dispersive X-ray spectroscopy analysis, neither amorphous layers nor Si impurity segregation were found at the grain boundary. The grain boundary blocking effect of the Sigma 5 boundary must be an intrinsic feature arising from its core structure and yttrium solute segregation of the grain boundary.